A common biological laboratory procedure is one involving examination and identification of microscopic single-cell organisms in the animal phylum Protozoa and the like. More than 30,000 protozoan species are known to exist and are divided into five classes based on their modes of locomotion and/or stationary condition. Such organisms live in fresh, brackish, or salt water or in moist organic debris or as parasites in the gut of larger animals. The living functions of a protozoan cell are performed by “organelles” which are pseudo or crude organs that execute the processes of locomotion, feeding, excretion, reproduction, and reaction to external stimuli. To observe, identify, and study protozoan organisms requires microscope magnification in the range of 40× to about 400×. For this purpose, the organisms of interest are cultivated in their normal habitat medium and the liquid cultures are transferred to a microscope stage for illuminated viewing. The liquid medium provides them with the nutrients necessary to live and to allow them to move about in normal routines.
To view such live and moving protozoans under microscope magnification, the liquid culture must be contained in a shallow reservoir that confines the protozoans to a liquid depth not much larger than the depth of field of the microscope and yet deep enough to allow the microscopic animals to move about freely in a normal way. The upper and lower parts of the reservoir must be optically clear to properly illuminate and view the protozoans and the culture medium must not contain an excess of bulk or suspended particles that can obscure the view. A variety of microscope slides have been developed to serve this purpose, with the simplest consisting of a two-component assembly comprising a glass slide with a shallow cavity ground into its upper surface and a cover glass, often referred to as a “coverslip”, to cover and enclose the liquid medium within the cavity. Other more elaborate arrangements have been developed to provide one or more thin liquid cavity within a molded clear plastic body in which the culture liquid is introduced as droplets from a syringe or pipette and allowed to intrude into and fill the cavity or cavities by capillary action.
In reference to two-component slides, Hall (U.S. Pat. No. 4,022,521) discloses a two-component microscope slide for examining biological samples such as blood cells which are typically less than 0.0005 inch in largest dimension. The base slide and coverslip components are molded from clear plastic material. Small micron-size projections or “posts” are molded on the upper surface of the base slide and are located in the liquid viewing area between the upper and lower slide components to preserve a uniform depth dimension of the liquid layer limited to not more than 15 μm (0.0006 inch) thick and open on all four sides. The small posts are spaced widely apart to minimize any obstruction of blood cells being freely dispersed into the thin liquid layer space. Berndt (U.S. Pat. No. 6,358,475) discloses a two-component microscope slide also intended for examining blood cells while intentionally allowing the coverslip component to sag downward into the liquid layer as a result of the capillary attraction force of the contained liquid in the thin layer acting on the flexible coverslip, thereby causing a non-uniform liquid layer thickness with the result that more cells reside in the thicker parts of the layer. Brackett (U.S. Pat. No. 3,551,023) discloses a two-component microscope slide in which the specimen to be viewed (preferably a pathology specimen) is centrally positioned between the base slide and a coverslip separated by a thin perimeter of material adhered to the base slide. The open area containing the specimen can be smaller than the base slide to minimize sagging of the coverslip. The perimeter spacer material is a two-sided pressure-sensitive adhesive film that allows the coverslip to be affixed to the base slide to completely encase the specimen. While some degree of control of the specimen layer thickness is provided by the adhesive film, this slide configuration is not necessarily intended for precise sample layer thickness control. If used to contain a liquid culture sample, this slide arrangement would be found to be ineffective in being filled without entrapping air bubbles and would introduce uncertainty in properly sealing the edges of the coverslip. Patterson (U.S. Pat. No. 3,447,863) discloses a method for preparing a microscope slide for viewing biological specimens in a liquid medium giving priority attention to providing a uniform specimen layer thickness. In this patent, the specimen is held within a prescribed liquid layer thickness by means of micro-sized polymer beads of a predetermined spherical diameter mixed into the specimen liquid medium. Such polymer beads having substantially uniform diameter are commercially available in a variety of sizes. For static biological specimens the positions and spacings of the beads does not present a significant problem with microscope viewing. However, for observing moving protozoans of say 0.003 inch size in a liquid layer 0.010 inch thick, the beads will be more than three times larger than the target living organisms and, hence, where clusters of beads form in the liquid layer the specimens of interest may become easily obscured from view.
In reference to integrally molded cavity slides, Golias (U.S. Pat. No. 4,505,557) discloses a multiple-chamber microscope slide molded as a one-piece plastic assembly. In this patent, the optically clear plastic base slide and coverslip viewing panels comprising each chamber are molded as a combined integral unit with a liquid chamber therebetween open on two ends and having an available liquid layer thickness dimension of 0.010 inch. This arrangement is generally useful for microscopic examination of biological liquids but it is intricate in its three-dimensional geometry, particularly where very thin plastic coverslip panels and thin liquid chamber dimensions are required. The thin integrally molded chambers and coverslip panels forming the multiple chambers of this slide require special molding techniques that make the slide fabrication time consuming and expensive. Parker (U.S. Pat. No. 4,299,441) discloses an optically clear multiple-chamber plastic molded microscope slide having its upper and lower specimen view panels combined as an integral unit with an internal liquid chamber open on one side and having a liquid layer thickness of 0.004 inch to 0.008 inch and an upper plastic viewing panel (“coverslip”) thickness of 0.013±0.002 inch. The internal cavity chambers having only one open side complicates the three-dimensional plastic molding process which, for thin plastic viewing panels and liquid layer cavity dimensions, can introduce inadvertent molding flaws or inaccurate dimensions in the multiple-chamber assembly. White (U.S. Pat. No. 3,961,346) discloses a liquid inspection slide for biological liquid specimens and cell cultures having thin specimen chambers open on one side molded in a unitary body of optically clear polymeric plastic material. The chambers are deliberately tapered in thickness at an angle of a few degrees resulting in a liquid layer thickness dimension ranging from about 0.005 inch on its closed end to about 0.015 inch on its open end. The plastic molding process prevents this layer thickness from being made smaller. However, the patent specifies that by applying pressure via small heated platens positioned on the coverslip and base slide panels the chamber thickness can be made noticeably smaller. Elkins (U.S. Pat. No. 3,777,283) discloses a microscope slide for liquid specimens having multiple thin liquid layer chambers molded in an integral clear plastic slide body. The liquid layer thickness is preferably 0.009 inch to 0.010 inch. The overall dimensions of this multi-chamber slide are 3 inches long by 1 inch wide by 0.0625 inch thick and has five individual specimen chambers approximately 0.375-inch wide. The plastic molding process for this slide demands skilled mold preparation and careful plastic injection control to produce accurate liquid chamber dimensions and thin chamber viewing panels without flaws. This patent is the forerunner to the more elaborate integrally molded microscope slide described above in Parker (U.S. Pat. No. 4,229,441).
Of particular concern in the prior art cited and described above is, in the two-component devices, the ability to readily achieve or use a very thin coverslip where emplaced rigidity is required to prevent sagging and, in the integrally molded devices, the complexity of the molding process required to achieve accurate dimensions and efficient production of the very thin walled three-dimensional cavity bodies. In particular, the thickness of the coverslip imposes a limitation on the degree of microscope magnification that can be used in viewing the protozoans in the liquid well or cavity. The optical path involves three different layers having different indices of refraction that can limit the microscope depth of field and focusing clarity of the objects being viewed. When the object is a live protozoan moving in the culture liquid layer, clear viewing can only be obtained under conventional conditions at magnifications up to about 40×. This degree of magnification does not permit detailed observation of the organelles and their internal functions within the cell body of small protozoans. However, common practice has shown that this magnification can be increased by a factor of two or three when using a standard microscope by placing a drop of oil between the closely spaced microscope objective lens and the coverslip to eliminate the air path. But no such improvements are possible when using digital microscopes that operate essentially in camera mode with a substantial air path. An alternative approach not requiring oil coupling is to make the coverslip thinner to minimize the intermediate plastic (or glass) segment of the optical path. This approach, although applicable to both optical and digital microscopes, has limitations because of the flexibility of a thin coverslip when required to maintain a uniform liquid layer thickness over a relatively wide span and in handling and emplacing such thin coverslip materials. In this alternative, however, only the coverslip component and not the base slide viewing panel must be made thin since the optical path associated with the microscope view is not significantly affected by the thickness of the base slide viewing panel. As another concern, molding plastic components that have a thin capillary liquid cavity and very thin viewing panels having the necessary microscope optical clarity for undistorted imaging is a demanding task requiring a high precision mold, skilled mold adjustments and readyness checking, and careful control of the plastic material injection process. Such demanding molding requirements and related quality control makes production of such three-dimensional cavity slide assemblies expensive and time consuming. For both types of liquid well microscope slides these requirements place a limit on the thinnest dimensions that can be achieved and used with reasonable production efficiency. Therefore, the liquid-culture microscope slide concepts and methodologies cited in the prior art limit the ability to magnify and observe with quantitative detail the many species of small protozoans of interest.
The microscope slide described hereinbelow circumvents the limitations concerning the relevant prior art by creating an improved two-component liquid-well slide configuration that facilitates the use of thin flexible plastic coverslips and requires straight-forward injection molding fabrication of only the plastic slide base component. This improved liquid-well slide assembly provides significant technical advantages in its manufacture and cost and important advances that allow the use of increased magnification for detailed observation and study of protozoans and other small cellular organisms.